Nucleic Acid-Drug Conjugate, Drug Delivery System, Preparation Method Therefor and Use Thereof

Abstract

Disclosed are a nucleic acid-drug conjugate based on a phosphorothioate-modified nucleic acid, a drug delivery system, and a preparation method therefor. The nucleic acid-drug conjugate is formed by reacting and conjugating a phosphorothioate group in a phosphorothioate-modified nucleic acid with a group that is modified on a drug molecule and able to undergo electrophilic reaction with the phosphorothioate groups; in addition, by selecting different nucleic acid sequences including functional nucleic acids, the nucleic acid-drug conjugate is able to be self-assembled into various forms of drug-containing nano-carriers for drug delivery.

1 . A nucleic acid-drug conjugate based on a phosphorothioate-modified nucleic acid, wherein comprising a phosphorothioate-modified nucleic acid skeleton and a drug molecule grafted onto the nucleic acid skeleton, and the grafting is realized by reacting the phosphorothioate groups on the nucleic acid skeleton with a group modified on the drug molecule that able to undergo electrophilic reaction with the phosphorothioate groups. 
     
     
         2 . The nucleic acid-drug conjugate according to  claim 1 , wherein on the phosphorothioate-modified nucleic acid skeleton, the sites and number of phosphorothioate modifications are able to be adjusted and controlled as required, the phosphorothioate group is continuously modified at one end of the nucleic acid sequence, and/or is selectively modified at the middle base sequence of the nucleic acid sequence, and the modification mode is multiple modification or single modification. 
     
     
         3 . The nucleic acid-drug conjugate according to  claim 1 , wherein the phosphorothioate-modified nucleic acid skeleton is prepared by a solid phase synthesis method. 
     
     
         4 . The nucleic acid-drug conjugate according to  claim 1 , wherein the sequence and segment type of the oligonucleotide of the phosphorothioate-modified nucleic acid skeleton are able to be independently designed, and a controllable DNA nanostructure is able to be further assembled through molecular recognition, and the nucleic acid-drug conjugate and the assembled structure thereof is able to be used as a novel drug delivery system. 
     
     
         5 . The nucleic acid-drug conjugate according to  claim 2 , wherein a phosphorothioate modification site is set every 2 to 3 bases on the nucleic acid skeleton of the nucleic acid used for the assembly of gel and tetrahedral structure; continuous phosphorothioate modifications at one end of a nucleic acid sequence are carried out on the nucleic acid used for micellar assembly to prepare block-type nucleic acids containing phosphodiester bonds and phosphorothioate bonds. 
     
     
         6 . The nucleic acid-drug conjugate according to  claim 1 , wherein a group that is able to undergo electrophilic reaction with phosphorothioate group is introduced into the drug molecule by a simple esterification or acylation reaction. 
     
     
         7 . The nucleic acid-drug conjugate according to  claim 1 , wherein a cleavable responsive chemical bond is further introduced into the drug molecule. 
     
     
         8 . The nucleic acid-drug conjugate according to  claim 1 , wherein the group that is able to modified on the drug molecule and undergo electrophilic reaction with phosphorothioate group is selected from one or more of the following: 1) a bromine-containing or iodine-containing functional group; 2) a maleimide group; 3) an aziridinyl sulfonamide group. 
     
     
         9 . The nucleic acid-drug conjugate according to  claim 1 , wherein the drug molecule is selected from one or more of anticancer drugs or cancer targeted drug molecules. 
     
     
         10 . The nucleic acid-drug conjugate according to  claim 1 , wherein the drug molecule grafted on the nucleic acid skeleton is a functional drug molecule, a fluorescent probe molecule, or a cell targeted molecule. 
     
     
         11 . The nucleic acid-drug conjugate according to  claim 1 , wherein the type of the nucleic acid skeleton is selected from a deoxyribonucleic acid sequence or a ribonucleic acid sequence; the sequence of the nucleic acid skeleton is selected one or more of:
 a non-functional common base sequence, including a simple nucleic acid sequence composed of one base and a complex nucleic acid sequence that is able to be used for precise structural assembly of nucleic acids;   a functional nucleic acid sequence, the functional nucleic acid sequence is selected from antisense nucleic acid sequence, nucleic acid aptamer sequence, nuclease sequence, small interfering RNA, messenger RNA, microRNA, long non-coding RNA, small hairpin RNA, guide RNA for gene editing, and circular RNA.   
     
     
         12 . The nucleic acid-drug conjugate according to  claim 1 , wherein the nucleic acid molecule grafted with the drug retains the property of base complementary pairing, by which the other functional nucleic acid sequences are paired to impart targeting and imaging functions to the nucleic acid-drug conjugate drug delivery system to prepare a multifunctional drug delivery system of nucleic acid-drug conjugate, wherein the functional nucleic acid for pairing is selected from nucleic acid aptamers, antisense nucleic acid sequences, fluorescent molecule-modified nucleic acid sequences, functional polypeptide-modified nucleic acid sequences, and targeted galactose-modified nucleic acid sequences. 
     
     
         13 . A drug delivery system, wherein the drug delivery system is a nano drug-loaded system formed by self-assembly of the nucleic acid-drug conjugate according to  claim 1 . 
     
     
         14 . The drug delivery system according to  claim 13 , wherein different drug delivery systems are able to be prepared by different methods according to the selected drug molecules and nucleic acid sequences, and the drug delivery systems are selected from simple chain nucleic acid-drug macromolecule drugs, precisely assembled DNA nanostructures, DNA nanogels, drug-loaded micellar spherical nucleic acid nanostructures, drug-loaded nucleic acid polyhedral structures, and drug-loaded nucleic acid hydrogels. 
     
     
         15 . The drug delivery system according to  claim 14 , wherein the drug molecules used to prepare the precisely assembled DNA nanostructures are selected from molecules with weak hydrophobicity and small molecular weight, and/or molecules with reduced number of phosphorothioate modifications on the nucleic acid skeleton; and the drug molecules used for micellar assembly are selected from highly hydrophobic molecules, and/or molecules with increased number of phosphorothioate modifications on the nucleic acid skeleton. 
     
     
         16 . A method for preparing the nucleic acid-drug conjugate according to  claim 1 , wherein comprising:
 step one, preparing a phosphorothioate-modified nucleic acid molecule;   preparing a drug molecule containing a group that is able to undergo electrophilic reaction with phosphorothioate group by a chemical reaction method;   there is no order among the steps of preparing the nucleic acid molecule and preparing the drug molecule;   step two, preparing a nucleic acid-drug conjugate, dissolving the modified drug molecule in an organic solvent, then adding an appropriate amount of nucleic acid molecule for the reaction, the drug molecule in the reaction is greatly excessive relative to the phosphorothioate groups, removing the excess small molecules after reaction, and drying to obtain the nucleic acid-drug conjugate.   
     
     
         17 . The method for preparing the nucleic acid-drug conjugate according to  claim 16 , wherein the phosphorothioate-modified nucleic acid molecule is prepared by a solid phase synthesis. 
     
     
         18 . The method for preparing the nucleic acid-drug conjugate according to  claim 16 , wherein the modification method of the drug molecule is selected from the following methods: (1) preparing carbonyl ethyl bromide drug molecules containing disulfide bonds through a two-step esterification reaction; and (2) preparing benzyl bromide structure-modified drug molecules containing disulfide bonds through a two-step esterification reaction. 
     
     
         19 . The method for preparing the nucleic acid-drug conjugate according to  claim 16 , wherein the drug molecule is further introduced with a cleavable responsive chemical bond, the cleavable responsive chemical bond is a disulfide bond, and the group that undergo electrophilic reaction with phosphorothioate groups is a bromo-modified group; the chemical reaction method for preparing a bromo-modified drug molecule containing disulfide bonds is selected from the following methods: introducing the disulfide bond into the camptothecin drug molecule through triphosgene under argon atmosphere, and then reacting with bromoacetyl bromide to obtain the camptothecin bromide drug molecule; and, carrying out an esterification reaction of 4-bromomethylbenzyl alcohol with dithiodipropylene to prepare the carboxylic acid structure containing disulfide bonds, and then carrying out an esterification reaction of the carboxylic acid with the 2′ hydroxyl group of paclitaxel to prepare the benzyl bromide structure-modified paclitaxel drug molecule. 
     
     
         20 . The method for preparing the nucleic acid-drug conjugate according to  claim 16 , wherein the grafting efficiency of drug molecules is able to be controlled by controlling the concentration of nucleic acid molecules, the ratio of drug molecules to phosphorothioate groups, and whether a salt solution is included in the reaction solution. 
     
     
         21 - 24 . (canceled)